Polysaccharide gel formulation having increased longevity

ABSTRACT

Described herein are polysaccharide gel formulations including at least one inhibitor of polysaccharide degradation and methods of making the same. The methods described herein involve the steps of providing at least one polysaccharide and incorporating at least one inhibitor of degradation into the polysaccharide. In some embodiments, the incorporating step comprises 1) mixing the at least one inhibitor with the at least one polysaccharide at a highly hydrated state thereby encapsulating the at least one inhibitor in a polysaccharide network, and 2) dehydrating the polysaccharide network thereby controlling release kinetics or final swell ratio. In another embodiment, the incorporating step comprises 1) encapsulating at least one inhibitor into a biocompatible or biodegradable vessel and 2) combining the polysaccharide and the vessel into a gel formulation. The polysaccharide gel formulations described herein can be used for a variety of cosmetic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/276,167, filed Nov. 21, 2008, which application claims the benefit ofU.S. provisional patent application No. 60/991,473, filed Nov. 30, 2007,the entire disclosures of which applications are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

Disclosed generally are formulations useful for increasingpolysaccharide gel longevity for cosmetic or medical applications, andrelated methods of making and using same.

BACKGROUND

Polysaccharides are relatively complex carbohydrates. They are polymersmade up of many monosaccharides joined together by glycosidic bonds.They are therefore large, often branched, macromolecules.Polysaccharides, especially hyaluronic acid (HA), have been useful incosmetic and medical applications. These polymers have been used, forexample, as fillers in soft tissue augmentation.

Residing in the extracellular space, HA functions as a space-filling,structure stabilizing, and cell protective molecule with uniquelymalleable physical properties and superb biocompatibility. HA matricesare extremely viscoelastic while preserving a high level of hydration. Astrong correlation exists between the water content in the skin andlevels of HA in dermal tissue. As human skin ages, there are knownalterations in HA content and metabolism. With these changes, there is asignificant deterioration in the mechanical properties of the skin.There appears to be a relationship between youthful skin and thepresence of a strong HA network in the intercellular matrix.

Unfortunately, non-cross-linked as well as cross-linked polysaccharidechains such as HA are subject to degradation through different pathways;(e.g. enzymatic, free radical) thus limiting the polymer's longevity invivo. It is, therefore, important to develop methods and compositionsthat decrease the rate of natural decomposition and increase theproduct's persistence in vivo. There remains an unmet need for having apolysaccharide formulation which has increased longevity by beingresistant to degradation.

SUMMARY

Described herein are polysaccharide gel (e.g. hyaluronic acid, HA)formulations with increased longevity, or increased degradation time, invivo. This increase in degradation time is provided by the incorporationof molecules that act as inhibitors to degradation. Further, the presentdisclosure provides methods for encapsulating these formulations tosustain a prolonged longevity or degradation time in vivo. The presentdisclosure also relates to the preparation of gels that can havepharmaceutical or cosmetic applications.

In one embodiment described herein is a polysaccharide gel formulationcomprising at least one polysaccharide selected from the groupconsisting of hyaluronic acid, cellulose, chitosan, o-sulfated HA,dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratinsulfate, heparin, heparin sulfate, and alginate, and at least oneinhibitor of polysaccharide degradation selected from the groupconsisting of a glycosaminoglycan, an antioxidant, a flavonoid, aprotein, a fatty acid, and combinations thereof. In one embodiment, thepolysaccharide is cross-linked. In one embodiment, the at least onepolysaccharide is hyaluronic acid.

In one embodiment, the glycosaminoglycan is selected from the groupconsisting of heparin, heparin sulfate, dermatan sulfate, chondroitinsulfate, o-sulfated hyaluronic acid, linamarin and amygdalin. In anotherembodiment, the glycosaminoglycan is chondroitin sulfate and is presentat a concentration of about 1% to about 40% by weight.

In one embodiment, antioxidant is selected from the group consisting ofascorbic acid, melatonin, vitamin C, vitamin E and combinations thereof.

In one embodiment, the flavonoid is selected from the group consistingof luteolin, apigenin, tangeritin, quercetin, kaemferol, myricetin,fisetin, isorhamnetin, pachypodol, rhamnazin, hesperetin, naringenin,eriodictyol, homoeriodictyol, taxifolin, dihydroquercetin,dihydrokaempferol, tannic acid, tannis, condensed tannis, hydrolysabletannis and combinations thereof. In another embodiment, the flavonoid istannic acid and is present at a concentration of about 0.0001 to about1% by weight.

In one embodiment, the protein is a serum hyaluronidase inhibitor. Inanother embodiment, the fatty acid is a saturated C₁₀₋₂₂ fatty acid.

In one embodiment, the formulation further comprises a biocompatible orbiodegradable vessel wherein the inhibitor is inside or part of saidvessel, wherein the vessel is a liposome, micelle, or polymerizedvesicle. In another embodiment, the inhibitor provides the formulationwith improved rheological properties resulting in less extrusion forcerequired for administration of the formulation compared to across-linked polysaccharide gel formulation.

In one embodiment described herein is a method of producing apolysaccharide gel formulation comprising having an increaseddegradation time comprising the step of: providing at least onepolysaccharide selected from the group consisting of hyaluronic acid,cellulose, chitosan, o-sulfated HA, dextran, dextran sulfate,chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparinsulfate, and alginate and incorporating in the polysaccharide at leastone inhibitor of polysaccharide degradation selected from the groupconsisting of a glycosaminoglycan, antioxidant, flavonoid, protein andfatty acid.

In one embodiment of the method, the incorporating step comprises 1)mixing the inhibitor with the polysaccharide at a highly hydrated statethereby encapsulating the inhibitor in a polysaccharide network, and 2)dehydrating the polysaccharide network thereby controlling releasekinetics or final swell ratio.

In another embodiment of the method, the at least one polysaccharide iscross-linked before incorporation of the at least one inhibitor ofpolysaccharide degradation using a cross-linker selected from the groupconsisting of 1,4-butanediol diglycidyl ether (BDDE),1,2-bis(2,3-epoxypropoxy)ethylene,1-(2,3-epoxypropyl)-2,3-epoxycyclohexane and combinations thereof.

In one embodiment of the method, the incorporating step comprises 1)encapsulating an inhibitor into a biocompatible or biodegradable vesseland 2) combining the at least one polysaccharide and the vessel into agel formulation.

In one embodiment described herein is a polysaccharide gel formulationwith an increased degradation time comprising cross-linked hyaluronicacid and tannic acid, wherein the tannic acid is present at aconcentration of about 0.0001% to about 1%.

In one embodiment described herein is a polysaccharide gel formulationwith an increased degradation time comprising cross-linked hyaluronicacid and chondroitin sulfate, wherein the chondroitin sulfate is presentat a concentration of about 1% to about 40%.

Further described herein are pharmaceutical compositions comprising apolysaccharide gel formulation, a pharmaceutically acceptable carrier,and an active ingredient. In one embodiment, the active ingredient isselected from the group consisting of anti-itch, anti-cellulite,anti-scarring, anti-inflammatory agents, antioxidants, vitamins,moisturizers and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically illustrates the extent of enzymatic degradation ofpolysaccharide gels including chondroitin sulfate A (CSA) using acalorimetric assay.

FIG. 2 graphically illustrates the extent of enzymatic degradation ofpolysaccharide gels including tannic acid (TA) using a colorimetricassay.

FIG. 3 graphically illustrates the extent of enzymatic degradation ofpolysaccharide gels both with and without CSA using a soluble HA assay.

FIG. 4 graphically illustrates the extent of enzymatic degradation ofpolysaccharide gels both with and without TA using a soluble HA assay.

FIG. 5 graphically illustrates effects of CSA on the extrusion force ofa polysaccharide gel.

DETAILED DESCRIPTION

Described herein are polysaccharide gel (e.g. hyaluronic acid, HA)formulations with increased longevity, or increased degradation time, invivo provided by incorporating molecules that act as inhibitors todegradation. In some embodiments, the polysaccharide gels arecross-linked. Further, the present disclosure also relates to methodsfor encapsulating these formulations to sustain a prolonged longevity ordegradation time in vivo. The present disclosure also relates to thepreparation of gels that can have pharmaceutical or cosmeticapplications.

One aspect of the present disclosure relates to polysaccharide gelformulations comprising at least one polysaccharide and at least oneinhibitor of polysaccharide degradation. The present disclosure furtherrelates to increasing the degradation time of polysaccharide gels byincorporating inhibitors to degradation. “Polysaccharide” refers to apolymer of more than two monosaccharide molecules, of which themonosaccharides can be identical or different. The polysaccharide of thepresent disclosure can be cross-linked or not cross-linked. Thepolysaccharides used herein can be, but are not limited to, HA,cellulose, chitosan, o-sulfated HA, dextran, dextran sulfate,chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparinsulfate, and alginate.

Inhibitors are molecules that act by targeting and neutralizing specificdegradation mechanisms such as enzymatic and free radical degradation.Molecules that display inhibitory activity include but are not limitedto glycosaminoglycans (GAGs), (e.g. heparin, heparan sulfate, dermatansulfate, chondroitin sulfate, o-sulfated HA, linamarin and amygdalin),antioxidants (e.g. ascorbic acid, melatonin, vitamin C and vitamin E),proteins (e.g. serum hyaluronidase inhibitor) and fatty acids (e.g.saturated C₁₀ to C₂₂ fatty acids).

Inhibitors are typically molecules orders of magnitude smaller thancross-linked polysaccharide polymers. Due to their small size and higherdiffusivity, they are prone to fast adsorption in vivo that couldpotentially limit their performance. One method of increasing the localhalf-life of such molecules in vivo is to chemically graft them to thepolysaccharide polymer network and deliver them simultaneously. Onedisadvantage of this method is that the bound molecule may displaysignificantly lower activity compared to the unbound one. In the presentpolysaccharide gel formulation, the inhibitor concentration can be inthe range of about 0.0001% to about 99% by wt, about 0.001% to about 75%by weight, about 0.01% to about 60% by weight, about 1% to about 50% byweight, about 1% to about 40% by weight, about 1% to about 30% byweight, about 1% to about 20% by weight, about 10% to about 20% byweight, about 20% to about 30% by weight 0.0001% to about 0.01% byweight, about 0.0001% to about 0.1% by weight, about 0.0001% to about 1%by weight, about 0.001% to about 1% by weight, about 0.01% to about 1%by weight, or about 1% to about 10% by weight.

Another aspect of the present disclosure relates to a method ofproducing a polysaccharide gel formulation having reduced degradationcomprising providing a polysaccharide and encapsulating an inhibitor ofdegradation in the polysaccharide.

Non-cross-linked as well as cross-linked polysaccharide chains aresubject to degradation through different pathways (e.g. enzymatic, freeradical) that often limits the polymer's longevity in vivo. It istherefore important to develop ways that decrease the rate of thisnatural decomposition process and increase the product's persistence intissues.

One method for achieving increased polysaccharide persistence is toencapsulate inhibitor molecules within the polysaccharide polymernetwork itself or into large vessels within the network that wouldenable local (injection site), sustained and controlled release ofdegradation inhibitors. This would also allow avoidance of the naturaldegradation mechanisms. The present encapsulation method provides aconstant supply of degradation inhibitors to the polysaccharide polymernetwork over a period of weeks. In other embodiments, a constant supplyof degradation inhibitors is provided over a period of months. Onemethod of encapsulation is to incorporate the degradation inhibitorswithin the polysaccharide polymer network either by adsorption or by anencapsulation process. In the latter case, the inhibitors are allowed tomix with the polysaccharide network at a highly hydrated state, followedby dehydration of the network to control the release kinetics (e.g.final swelling ratio of the polymer). A highly hydrated statecorresponds to an HA concentration that is less than about 20 mg/ml.

The final swelling ratio can be controlled by adjusting the pH orpartially dehydrating the polysaccharide network. The contracted networkcan be sized into particles, mixed with the polysaccharide gel anddelivered at the site of the injection. The slow re-hydration of theinhibitor-loaded polysaccharide particles can provide a sustained andcontrolled delivery of their active content.

Another aspect of the present disclosure relates to a polysaccharide gelformulation comprising a polysaccharide and an inhibitor ofpolysaccharide degradation, and further comprising a biocompatible orbiodegradable vessel wherein the inhibitor is inside or part of thevessel. Such vessels can be composed of non-covalently or covalentlylinked self-assembled molecules such as liposomes, micelles, andpolymerized vesicles.

A liposome is a vesicle composed of one or more bilayer membranes formedof naturally-derived phospholipids with mixed lipid chains (such as eggphosphatidylethanolamine), or of pure surfactant components likedioleoylphosphatidylethanolamine (DOPE). Liposomes, usually but not bydefinition, contain a core of aqueous solution; lipid structures thatcontain no aqueous material are called micelles. A micelle is anaggregate of surfactant molecules dispersed in a liquid colloid. Atypical micelle in aqueous solution forms an aggregate with thehydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic “tail” regions in the micelle center. Thistype of micelle is known as a normal phase micelle (oil-in-watermicelle). Inverse micelles have the headgroups at the centre with thetails extending out (water-in-oil micelle). Micelles are oftenapproximately spherical in shape, however, other forms, including shapessuch as ellipsoids, cylinders, and bilayers are also possible. The shapeand size of a micelle is a function of the molecular geometry of itssurfactant molecules and solution conditions such as surfactantconcentration, temperature, pH, and ionic strength. The process offorming micelles is known as micellization and forms part of the phasebehavior of many lipids according to their polymorphism.

Another aspect of the present disclosure relates to a method forproducing a polysaccharide gel formulation having reduced degradationcomprising the steps of 1) providing a polysaccharide, 2) incorporatingan inhibitor into a biocompatible or biodegradable vessel and 3)combining said polysaccharide and vessel into a gel formulation. Thismethod of encapsulation thus incorporates the inhibitors intobiocompatible and biodegradable vessels that could be delivered at thesame time with the polysaccharide. Such vessels can be composed ofnon-covalently or covalently linked self-assembled molecules (e.g.micelles, liposomes, and polymerized vesicles). Self-assembly is a termused herein to describe processes in which a disordered system ofpre-existing components forms an organized structure or pattern as aconsequence of specific, local interactions among the componentsthemselves, without external direction.

An additional advantage of the proposed formulation is the increasedtune-ability of the final product's rheological properties. Cross-linkedpolysaccharide gels typically have high viscosity and requireconsiderable force to extrude through a fine needle. Uncross-linkedpolysaccharides are often used as lubricants to facilitate thisextrusion process. However, especially in HA dermal fillers,uncross-linked HA does not contribute to the persistence of the finalproduct in vivo. In fact, the more cross-linked HA is replaced byuncross-linked HA to tune the rheological properties of the dermalfiller (for a fixed total HA concentration), the lower the degradationresistance of the product will be. Instead, according to the proposedformulation, uncross-linked GAGs that are also inhibitors to degradation(e.g. chondroitin sulfate, o-sulfated hyaluronic acid) can be used bothto extend the longevity and improve the rheological properties of thefinal product.

The polysaccharides described herein can be cross-linked oruncross-linked. A cross-linking agent can be used to cross-link thepolysaccharides according to the present description. The cross-linkingagent may be any agent known to be suitable for cross-linkingpolysaccharides and their derivatives via their hydroxyl groups.Suitable cross-linking agents include but are not limited to, forexample, 1,4-butanediol diglycidyl ether (or1,4-bis(2,3-epoxypropoxy)butane or 1,4-bisglycidyloxybutane, all ofwhich are commonly known as BDDE), 1,2-bis(2,3-epoxypropoxy)ethylene and1-(2,3-epoxypropyl)-2,3-epoxycyclohexane. The use of more than onecross-linking agent or a different cross-linking agent is not excludedfrom the scope of the present disclosure. In one embodiment, thecross-linking agent comprises or consists of BDDE.

Dermal fillers can be used to treat moderate to severe facial wrinklesand folds such as nasolabial folds (those lines that extend from thenose to the corners of the mouth). Dermal fillers can be a gel implantthat includes HA, a natural complex sugar that bolsters skin elasticity,providing a smooth and supple appearance. It is biocompatible and cansupplement the body's natural HA, which aging can deplete.

A dermal gel can be injected with a syringe into the mid to deep dermisof the face. The dermis is the subsurface skin layer that containsconnective tissue, nerve endings, sweat and oil glands, and bloodvessels. Dermal fillers can improve the skins appearance by lifting andadding volume to the wrinkles and folds in the treatment area.

Another aspect of the present disclosure relates to a cosmeticcomposition comprising the present polysaccharide gel formulation, acosmetic carrier, and an active ingredient. The cosmetic activeingredients may include but are not limited to antioxidants, vitamins,and moisturizers.

As used herein, “cosmetic” is an adjective referring to improving theappearance of a surface or covering defects. Typically, cosmeticcompositions can be used to improve aesthetic rather than functionalaspects of a surface. Most commonly, cosmetic compositions areformulated for application as a health and beauty treatment or foraffecting personal appearance of the body, for example, keratinoussurfaces such as skin, hair, nails, and the like.

As used herein, “cosmetically acceptable carrier” refers to a materialwhich is suitable for application to keratinous surfaces or other areasof the body. Upon application, cosmetically acceptable carriers aresubstantially free of adverse reactions with skin and other keratinoussurfaces. For example, the cosmetic carriers may take the form of fattyor non-fatty creams, milky suspensions or emulsion-in-oil oroil-in-water types, lotions, gels or jellies, colloidal or non-colloidalaqueous or oily solutions, pastes, aerosols, soluble tablets or sticks.

As used herein, “formulation” and “composition” may be usedinterchangeably and refer to a combination of elements that is presentedtogether for a given purpose. Such terms are well known to those ofordinary skill in the art.

As used herein, “carrier,” “inert carrier,” and “acceptable carrier” maybe used interchangeably and refer to a carrier which may be combinedwith the presently disclosed polysaccharide gel in order to provide adesired composition. Those of ordinary skill in the art will recognize anumber of carriers that are well known for making specific remedialpharmaceutical and/or cosmetic compositions.

Another aspect of the present disclosure relates to a pharmaceuticalcomposition comprising the polysaccharide gel formulation, apharmaceutical carrier and an active ingredient. As used herein, anactive ingredient includes but is not limited to a drug. A drug cangenerally be defined as a chemical substance used in the treatment,cure, prevention, or diagnosis of disease or used to otherwise enhancephysical or mental well-being.

Examples of active ingredients which can be included in the presentpharmaceutical composition are anti-itch, anti-cellulite, anti-scarring,and anti-inflammatory agents. Anti-itch agents can include methylsulphonyl methane, sodium bicarbonate, calamine, allantoin, kaolin,peppermint, tea tree oil and combinations thereof. Anti-cellulite agentscan include forskolin, xanthine compounds such as, but not limited to,caffeine, theophylline, theobromine, and aminophylline, and combinationsthereof. Anti-scarring agents can include IFN-γ, fluorouracil,poly(lactic-co-glycolic acid), methylated polyethylene glycol,polylactic acid, polyethylene glycol and combinations thereof.Anti-inflammatory agents can include dexamethasone, prednisolone,corticosterone, budesonide, estrogen, sulfasalazine, mesalamine andcombinations thereof.

A polysaccharide such as HA is found naturally in many tissues of thebody, such as, but not limited to, skin, cartilage, and the vitreoushumor. It is therefore well suited to biomedical applications targetingthese tissues. HA can be used in eye surgery (i.e., cornealtransplantation, cataract surgery, glaucoma surgery and surgery torepair retinal detachment). HA is also used to treat osteoarthritis ofthe knee. Such treatments, called visco-supplementation, areadministered as a course of injections into the knee joint and arebelieved to supplement the viscosity of the joint fluid, therebylubricating the joint, cushioning the joint, and producing an analgesiceffect. It has also been suggested that HA has positive biochemicaleffects on cartilage cells. Oral use of HA has been lately suggested,although its effectiveness needs to be demonstrated. At present, thereare some preliminary clinical studies that suggest that oraladministration of HA has a positive effect on osteoarthritis.

Due to its high biocompatibility and its common presence in theextracellular matrix of tissues, HA can be used as a biomaterialscaffold in tissue engineering research. In some cancers, HA levelscorrelate well with malignancy and poor prognosis. HA is thus often usedas a tumor marker for prostate and breast cancer. It may also be used tomonitor the progression of the disease. HA may also be usedpostoperatively to induce tissue healing, notably after cataractsurgery. Current models of wound healing propose that larger polymers ofHA appear in the early stages of healing to physically make room forwhite blood cells, which mediate the immune response.

A pharmaceutical composition can optionally include one or more agentssuch as, without limitation, emulsifying agents, wetting agents,sweetening or flavoring agents, tonicity adjusters, preservatives,buffers antioxidants and flavonoids. Tonicity adjustors useful in apharmaceutical composition of the present disclosure include, but arenot limited to, salts such as sodium acetate, sodium chloride, potassiumchloride, mannitol or glycerin and other pharmaceutically acceptabletonicity adjusters. Preservatives useful in the pharmaceuticalcompositions described herein include, without limitation, benzalkoniumchloride, chlorobutanol, thimerosal, phenyl mercuric acetate, and phenylmercuric nitrate. Various buffers and means for adjusting pH can be usedto prepare a pharmaceutical composition, including but not limited to,acetate buffers, citrate buffers, phosphate buffers and borate buffers.Similarly, antioxidants useful in pharmaceutical compositions are wellknown in the art and includes for example, sodium metabisulfite, sodiumthiosulfate, acetylcysteine, butylated hydroxyanisole and butylatedhydroxytoluene. Flavonoids are compounds found in plants that are wellknown to have diverse beneficial biochemical and antioxidant effects.Subcategories of flavonoids include: flavones, flavonols, flavanonse andflavanonols. Examples of flavonoids include: luteolin, apigenin,tangeritin, quercetin, kaempferol, myricetin, fisetin, isorhamnetin,pachypodol, rhamnazin, hesperetin, naringenin, eriodictyol,homoeriodictyol, taxifolin, dihydroquercetin, dihydrokaempferol, tannicacid, tannis, condensed tannis, and hydrolysable tannis. It isunderstood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition of theinvention. See for example, Remington's Pharmaceutical Sciences MacPublishing Company, Easton, Pa. 16^(th) Edition 1980.

Some of the advantages of the invention are illustrated below usingexamples which describe the preparation of an HA filling gel accordingto the methods described herein, the preparation of an HA filling gelaccording to the prior art and the degradation tests performed on thosesamples.

Molecular weight (M_(w)) as used herein refers to the sum of the atomicweights of the atoms in a molecule. For example, that of methane (CH₄)is 16.043 g/mol, the atomic weights being carbon=12.011 g/mol,hydrogen=1.008 g/mol. A Dalton (Da) is a unit of mass equal to 1/12 themass of ¹²O and one million Da can be notated as 1 MDa.

EXAMPLE 1 Preparation of a HA Filling Gel According to the PresentDisclosure

One to five grams of polysaccharide filler with a HA concentration of 24mg/mL, about a 6% degree of cross-linking and a G′ of about 180(JUVÉDERM® 24HV, (Allergan Inc., Irvine, Calif.)) are mixed with 1000 μlof a phosphate buffered saline (PBS) solution (pH˜7) that issupplemented with 10-200 mg of chondroitin sulfate A(CSA−M_(w)=5,000-120,000 Da). The mixture is mechanically homogenized.

EXAMPLE 2 Preparation of a HA Filling Gel by the Process of the PriorArt

One to five grams of polysaccharide filler with a HA concentration of 24mg/mL, about a 6% degree of cross-linking and a G′ of about 180(JUVÉDERM® 24HV) are mixed with 1000 μl of PBS such that the final HAconcentration is the same as in Example 1. The mixture is mechanicallyhomogenized.

EXAMPLE 3 An Alternative Preparation of a Hyaluronic Acid Filling GelAccording to the Present Disclosure

One to five grams of a HA based polysaccharide filler with a HAconcentration of 24 mg/mL, about a 6% degree of cross-linking and a G′of about 170 (JUVÉDERM® 30) are mixed with 50 μl of PBS solution (pH˜7)that is supplemented with 1-20 mg of tannic acid (TA−M_(w)=800-4,000Da). The mixture is mechanically homogenized.

EXAMPLE 4 An Alternative Preparation of a Hyaluronic Acid Filling Gel bythe Process of the Prior Art

One to five grams of a HA based polysaccharide filler with a HAconcentration of 24 mg/mL, about a 6% degree of cross-linking and a G′of about 170 (JUVÉDERM® 30) are mixed with 50 μl of PBS such that thefinal HA concentration is the same as in Example 3. The mixture ismechanically homogenized.

EXAMPLE 5 Preparation of a Hyaluronic Acid Filling Gel According to thePresent Disclosure

One gram of sodium hyaluronate fibers (NaHA, M_(w)=0.5-3 MDa) is mixedwith 5-10 g of 1% sodium hydroxide solution and the mixture is left tohydrate for 1-5 hrs.

Fifty to two hundred milligrams of 1,4-butanediol diglycidyl ether(BDDE) are added to the NaHA gel and the mixture is mechanicallyhomogenized.

The mixture is then placed in a 40-70° C. oven for 1-4 hrs.

The resulting cross-linked hydrogel is neutralized with an equimolaramount of hydrochloric acid (HCl) and swelled in PBS (pH=7).

Ten to two hundred milligrams of CSA (M_(w)=5,000-120,000 Da) are addedand the hydrogel is mechanically homogenized.

EXAMPLE 6 Preparation of an Hyaluronic Acid (HA) Filling Gel by theProcess of the Prior Art

One gram of NaHA (M_(w)=0.5-3 MDa) is mixed with 5-10 g of 1% sodiumhydroxide solution and the mixture is left to hydrate for 1-5 hrs.

Fifty to two hundred milligrams of BDDE (same HA: cross-linker molarratio as in Example 5) are added to the NaHA gel and the mixture ismechanically homogenized.

The mixture is then placed in a 40-70° C. oven for 1-4 hrs.

The resulting cross-linked hydrogel is neutralized with an equimolaramount of hydrochloric acid (HCl) and swelled in PBS (pH=7) such thatthe final HA concentration is the same as in Example 5. The obtainedhydrogel is mechanically homogenized.

EXAMPLE 7 Enzymatic Degradation Study (Colorimetric Test)

Resistance to enzymatic degradation of the HA filling gels prepared inExamples 1 and 2, was evaluated using the Morgan-Elson colorimetricassay. This assay is used to estimate the average molecular weight ofthe HA chains before and after enzymatic degradation.

Hyaluronidase (0.1-10 mg) was added to the HA samples for 10-250 mins at37° C. followed by 0.1 ml of a 0.8 M potassium tetraborate solution andheating at 100° C. for 10 mins. The samples were supplemented with 3 mlof a 10% (wt) p-dimethylaminobenzaldehyde solution in acetic acid andincubated at 37° C. for 10-120 mins. The change in the optical density(OD) at 585 nm post and pre-degradation was used to quantify the extentof degradation in each sample.

The results of the measurements made on the filling gels preparedaccording to the methods of the present disclosure and according to theprior art shown in FIG. 1 (Enzymatic Degradation Test Results(Colorimetric Assay)) indicate that the OD values of the gel prepared bythe methods of the present disclosure (Example 1: 0.774-25.184 mg/mlCSA) are lower than that of the gel prepared by the process of the priorart (Example 2: 0 mg/ml CSA). Furthermore, the decrease in the OD valuesis proportional to the concentration of CSA. Since the OD valuerepresents the extent of degradation, the results suggest that the gelsprepared according to the present methods display a 3-75% higherenzymatic degradation resistance than the gel prepared according to theprior art.

Similarly to the case of the CSA supplemented gels, the OD values of theTA supplemented gels prepared by the methods of the present disclosure,as shown in FIG. 2, (Example 3: 0.063-1.000 mg/mL TA) are lower thanthat of the gel prepared by the method of the prior art (Example 4: 0mg/mL TA). Furthermore, the decrease in the OD values is proportional tothe concentration of TA. Since the OD value represents the extent ofdegradation, the results suggest that the gels prepared according to thepresent description display a 15-90% higher enzymatic degradationresistance than the gel prepared according to the prior art. It canfurther be seen that TA has a higher inhibitory activity than CSA sinceit generally takes an order of magnitude less amount of TA to obtain thesame inhibition as with CSA.

EXAMPLE 8 Enzymatic Degradation Study (Soluble HA Assay)

To further evaluate the enzymatic degradation resistance of the HAfilling gels prepared in Examples 1 and 2, a SEC-MALS (Size ExclusionMulti-Angle Light Scattering) based soluble HA assay was used. Thisassay can be used to quantify degradation by evaluating the percentageof soluble HA (defined as the portion of the gel that can pass through a0.2-1.0 μm filter) contained in each sample. The change in the amount ofsoluble HA, pre and post-degradation, can be used to quantify the extentof degradation in each sample.

The SEC-MALS tests were performed using an Agilent size exclusionchromatography system equipped with Wyatt light scattering andrefractive index units. Hyaluronidase (0.1-10 mg) was added to the HAsamples for 10-250 mins at 37° C. followed by 0.1 ml of a 0.8 Mpotassium tetraborate solution and heating at 100° C. for 10 mins. Thesamples were diluted in PBS, filtered through a 0.2-1.0 μm filter andinjected into the SEC-MALS system. The soluble HA content before andafter the enzymatic degradation as well as their difference are shown inFIG. 3.

The results shown in FIG. 3 (Enzymatic Degradation Test Results(SEC-MALS Assay)) indicate that the increase in the soluble HA contentpost enzymatic degradation is significantly greater for the non-CSAcontaining sample. This difference in the soluble HA increase betweenthe CSA and the non-CSA samples is consistent with the results obtainedin the colorimetric degradation assay and suggests that the gel preparedaccording to the methods of the present disclosure shows a higherdegradation resistance than the gel prepared according to the prior art.

Similarly to the case of the CSA supplemented gels, the post degradationincrease in the soluble HA content of the TA supplemented gels preparedby the methods of the present description (Example 3: 0.063-1.000 mg/mLTA) is lower than that of the gel prepared by the process of the priorart (Example 4: 0 mg/mL TA). These results are in agreement with thecolorimetric method summarized in FIG. 4. Furthermore, it can be seenagain that the inhibitory activity of TA is an order of magnitude higherthan that of CSA.

EXAMPLE 9 Continuous Extrusion Force Test

To evaluate the rheological properties of the hyaluronic acid fillinggels prepared in Examples 5 and 6 continuous extrusion force tests wereperformed on each sample. The extrusion force test can be used todetermine whether CSA can facilitate the extrusion process, by acting asa lubricant.

The extrusion force tests were performed on an Instron instrument usinga 5 ml syringe with a 30G needle. 0.5 ml of each sample was extruded ata constant rate of 50 mm/min. The peak force recorded quantifies theease of extrusion. The compressive force as a function of thecompressive extension for the two samples is plotted in FIG. 5.

The results in FIG. 5 show that the extrusion force recorded for the gelprepared by the methods described herein is lower than that of the gelprepared by the process of the prior art. This difference in theextrusion force is characteristic of the difference in gel hardnessunder flow and suggests that the CSA contained in the gel prepared bythe methods described herein acts as a lubricant that facilitates flow.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or and consisting essentially of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

We claim:
 1. A dermal filler formulation comprising: (a) at least onepolysaccharide comprising crosslinked hyaluronic acid; (b) at least oneinhibitor of polysaccharide degradation, the inhibitor comprising tannicacid present in formulation at a concentration of about 0.0001% to about1% by weight; and (c) mannitol.
 2. The formulation of claim 1 furthercomprising vitamin C.
 3. The formulation of claim 1, wherein the atleast one polysaccharide is in the form of a biodegradable vesselwherein said inhibitor is encapsulated in said vessel.
 4. Theformulation of claim 1 wherein said at least one polysaccharide furthercomprises a chondroitin sulfate.
 5. The formulation of claim 1 whereinthe at least one polysaccharide is in the form of polysaccharide polymernetwork and the inhibitor is incorporated into the network while thenetwork is in a hydrated state.